Structural and regulatory studies on protein components of the E. coli sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) continued. The first component of the PTS (enzyme I, EI) is phosphorylated by phosphoenolpyruvate (PEP) on an active site histidine in a Mg(2+)-requiring reaction to produce pyruvate. New studies, in collaboration with Ann Ginsburg (see Reference 3), utilized the inactive mutant of EI(H189A), in which alanine is substituted for the active site His189, where substrate binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP strongly promotes dimerization of EI(H189A), 5 mM pyruvate has the opposite effect. When the coupling between N- and C-terminal domain unfolding produced by PEP and Mg(2+)is inhibited by pyruvate, the dimerization constant for EI(H189A) decreases from >10 to the eighth power to <5x10 to the fifth power. PEP binds to one site/monomer of EI(H189A); this binding is synergistic with Mg++. The results of this study show that physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-EI. In collaboration with the laboratories of Clore (NIH) and Wang (Omaha), solution structures by NMR of components of the PTS have continued. The general phosphocarrier HPr interacts with numerous sugar-specific IIA components, The structure of the complex between HPr and the cytoplasmic domain of the IIA for mannitol was solved (see Reference 2). A convex surface on HPr, formed by helices 1 and 2, interacts with a complementary concave depression on the IIA surface formed by helix 3, portions of helices 2 and 4, and beta strands 2 and 3. The majority of intermolecular contacts are hydrophobic, with a small number of electrostatic interactions at the periphery of the interface. The active site histidines of HPr and IIA are in close proximity so that a pentacoordinate phosphoryl transition state can be readily formed with only minimal perturbation of the backbone. Comparison with our two previously solved structures of HPr with partner proteins (enzyme I and the IIA for glucose) shows common features despite absence of structural resemblances. Consequently, different underlying structural elements can form binding surfaces for HPr that are similar in both shape and residue composition. The structure of the complex between the IIA for glucose and the cytoplasmic domain of IICB for glucose was solved (see Reference 1). The interface is formed by interaction of a concave depression on IIA with a concave protrusion on IIB; the phosphoryl donor and acceptor residues are in close proximity and buried at the center of the interface. Hydrophobic intermolecular contacts are supplemented by peripheral electrostatic interactions involving an alternating distribution of positively and negatively charged residues on the interaction surfaces. A phosphoryl transition state is easily accommodated without any change in backbone conformation and the structure of the complex accounts for the directionality of phosphoryl transfer between IIA nd IIB. The N-terminal domain of glucose IIA confers amphitropism to the protein, allowing it to shuttle between the membrane and cytoplasm. The structure of a synthetic peptide corresponding to the N-terminal domain was solved (see Reference 6). In water, the structure is disordered, but in detergent micelles, residues Phe3-Val10 of the peptide adopt a helical conformation. Of the four lysines in the N-terminal domain, only Lys5 and Lys7 in the amphipathic region interact with detergent. Intermolecular NOEs from detergent to the peptide were shown, supporting an anchor function to the membrane for the N-terminal domain. The enzyme IIA for glucose plays a direct role in regulating lactose permease; dephosphorylated IIA binds directly to the permease in the presence of a galactoside substrate. In collaboration with the Kaback laboratory lab (see reference 5),a double Cys mutation (Ile129Cys/Lys131Cys) was introduced into helix IV of the permease near the IIA binding site in cytoplasmic loop IV/V and in the vicinity of the substrate binding site at the interfaces of helices IV, V and VIII. The mutant no longer requires substrate for IIA binding; this mutant binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane. It is suggested that the double mutant is locked in an inward-facing conformation. A calmodulin-like protein from Mycobacterium smegmatis was purified to homogeneity and partially sequenced, in collaboration with the Reddy lab (see Reference 4); these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino acid open reading frame. The M. smegmatis protein, expressed in E. coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin anatagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids in the protein, there is one putative calcium-binding domain, compared to four such domains for eukaryotic calmodulin, reflecting the smaller size (~ 6 kDa) of this protein. Ultracentrifugal and mass spectral analysis excluded the possibility that calcium promotes oligomerization of the purified protein.